Summary
Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes.
Competing Interest Statement
JKM, LTG, EEH, HZ, BT, EL, JTT, and BPL are inventors on several U.S. provisional patent applications related to this work. All authors declare no other competing interests.
Footnotes
# Lead contact.
In this revision, we have drastically increased the number of enhancer-AAV vectors we tested and validated in mouse neocortex, and also expanded the scope with several well-validated vectors in non-human primate and in human ex vivo slice culture. We think several of these vectors are ready for wider deployment as research tools for non-species-restricted study of neurons, including collections of enhancer-AAV vectors targeting the PVALB and LAMP5 subclasses of neocortical interneurons. These results also gave us valuable insights into the best strategies for efficiently finding and building and screening enhancer-AAV vectors which we describe in detail. As a result, this revision is substantially increased in scope and improved in quality compared to the original.